Voltage-activated, CIC chloride (CI-) channels are essential for survival as illustrated by various inherited human diseases and knockout mouse models. Genetic mutations of distinct members of the CIC gene family lead to either impaired transepithelial ion transport in Bartter's syndrome, to increased muscle excitability in myotonia congenita, to reduced endosomal acidification and endocytosis in Dent's disease, or to impaired extracellular acidification by osteoclasts in osteopetrosis. Moreover, targeted disruption of several CIC channel genes in mice results in blindness. The three-dimensional structure of bacterial CIC channels determined by X-ray analysis provides the structural framework needed to perform structure-function analysis of CIC channel proteins in order to understand their biophysical properties, including the mechanism by which these channels are activated. This project will determine the gating and modulation of the CIC-2 CI-channel cloned from mouse parotid acinar cells when expressed in human kidney cells. We will determine if fast and slow gating processes control the kinetics of CIC-2, as shown for CIC-0 and CIC-1. Next, we will investigate how the large changes in intracellular and extracellular [CI-] and [H+] that epithelial cells undergo affect gating. We hypothesize that these changes serve as feedback signals that regulate channel activity as follows: chloride ions, by serving as a charge provider, regulate the voltage sensitivity of gating; and protons regulate channel activity by interacting directly with the gating machinery of the CIC-2 channel. Thus, we propose that the identification of the H+ binding sites will define the structural determinants of CIC-2 gating. The experiments proposed in Aim 1 will study the gating of CIC-2 and identify amino acids that form the gates of the channel. Aims 2 and 3 are designed to define the effects of CI- and H+ on CIC-2 CI- channel gating, as well as the molecular domains conferring voltage and pH sensitivity with the ultimate goal of defining the structure(s) responsible for gating. A combination of electrophysiological, molecular biology and amino acid-labeling techniques will be used to perform structure-function analysis of the channel protein and to track changes in gating, CI- and H+ sensitivity. This research will be carried out primarily at Universidad Autonoma de San Luis Potosi, Mexico in collaboration with Dr. Jorge Arreola as an extension of NIH grant # R01 DE09692.